Anti-EGFR antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to EGFR and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human EGFR with high affinity. In certain embodiments, the antibodies of the present invention are capable of inhibiting the growth of tumor cells expressing high levels of EGFR and/or inducing antibody-dependent cell-mediated cytotoxicity (ADCC) of such cells. The antibodies of the invention are useful for the treatment of various cancers as well as other EGFR-related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of USprovisional application Nos. 61/663,984, filed on Jun. 25, 2012; and61/821,000, filed on May 8, 2013, the disclosures of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for human EGFR, and methods of usethereof.

BACKGROUND

Epidermal growth factor receptor (EGFR, also known as HER1 or ErbB1) isa member of the ErbB/HER family of type 1 receptor tyrosine kinases(RTKs). Other members of this family include ErbB2 (HER2 or Neu), ErbB3(HER3) and ErbB4 (HER4). Known ligands for EGFR include epidermal growthfactor (EGF) and transforming growth factor alpha (TGF-α). Ligandbinding to EGFR induces tyrosine phosphorylation and receptordimerization with other ErbB family members.

RTKs such as EGFR function to allow cells to respond to diverse externalstimuli. However, aberrant activation and/or overexpression of EGFR isassociated with the development and progression of several humancancers. Accordingly, EGFR is a target for anti-cancer therapies.Approved drugs targeting EGFR include small molecule inhibitors such asgefitinib (Iressa®) and erlotinib (Tarceva®), and anti-EGFR antibodiessuch as cetuximab (Erbitux®) and panitumumab (Vectibix®). Anti-EGFRantibodies are mentioned in, e.g., U.S. Pat. No. 4,943,533, U.S. Pat.No. 5,844,093, U.S. Pat. No. 7,060,808, U.S. Pat. No. 7,247,301, U.S.Pat. No. 7,595,378, U.S. Pat. No. 7,723,484, and U.S. Pat. No.7,939,072. Nonetheless, there is a need in the art for novel EGFRantagonists, such as anti-EGFR antibodies, for the treatment of cancerand other related disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind human EGFR. Theantibodies of the invention are useful, inter alia, for inhibitingEGFR-mediated signaling and for treating diseases and disorders causedby or related to EGFR activity and/or signaling. The antibodies of theinvention are also useful for inducing cell death in cells that expresshigh levels of EGFR on their surfaces.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

The present invention provides antibodies, or antigen-binding fragmentsthereof comprising a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258,274, 290, 306, 322, 338, 354, and 370, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, 314, 330, 346, 362, and 378, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 322/330, 338/346, 354/362, and 370/378.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,232, 248, 264, 280, 296, 312, 328, 344, 360, and 376, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224,240, 256, 272, 288, 304, 320, 336, 352, 368, and 384, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208,216/224, 232/240, 248/256, 264/272, 280/288, 296/304, 312/320, 328/336,344/352, 360/368, and 376/384.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260,276, 292, 308, 324, 340, 356, and 372, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 6,22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246,262, 278, 294, 310, 326, 342, 358, and 374, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a light chain CDR1 (LCDR1) domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 12,28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252,268, 284, 300, 316, 332, 348, 364, and 380, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a light chain CDR2 (LCDR2) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222,238, 254, 270, 286, 302, 318, 334, 350, 366, and 382, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H1M085N);20-22-24-28-30-32 (e.g. H1M086N); 36-38-40-44-46-48 (e.g. H1M089N);52-54-56-60-62-64 (e.g. H1M102N); 68-70-72-76-78-80 (e.g. H1M103N);84-86-88-92-94-96 (e.g. H1M116N); 100-102-104-108-110-112 (e.g.H1H134P); 116-118-120-124-126-128 (e.g. H1H136P);132-134-136-140-142-144 (e.g. H1H141P); 148-150-152-156-158-160 (e.g.,H1H142P); 164-166-168-172-174-176 (e.g. H1H143P);180-182-184-188-190-192 (e.g., H1H144P); 196-198-200-204-206-208 (e.g.H1H145P); 212-214-216-220-222-224 (e.g. H1H147P);228-230-232-236-238-240 (e.g. H1H151P); 244-246-248-252-254-256 (e.g.H1H153P); 260-262-264-268-270-272 (e.g. H1H155P);276-278-280-284-286-288 (e.g. H1H157P); 292-294-296-300-302-304 (e.g.H1H158P); 308-310-312-316-318-320 (e.g. H1H159P);324-326-328-332-334-336 (e.g. H1H161P); 340-342-344-348-350-352 (e.g.H1H163P); 356-358-360-364-366-368 (e.g. H1H169P); and372-374-376-380-382-384 (e.g. H1H171P).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds EGFR,wherein the antibody or fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266,274/282, 290/298, 306/314, 322/330, 338/346, 354/362, and 370/378.Methods and techniques for identifying CDRs within HCVR and LCVR aminoacid sequences are well known in the art and can be used to identifyCDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In another aspect, the invention provides nucleic acid moleculesencoding anti-EGFR antibodies or antigen-binding fragments thereof.Recombinant expression vectors carrying the nucleic acids of theinvention, and host cells into which such vectors have been introduced,are also encompassed by the invention, as are methods of producing theantibodies by culturing the host cells under conditions permittingproduction of the antibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337,353, and 369, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofcomprising a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361,and 377, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247,263, 279, 295, 311, 327, 343, 359, and 375, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof; and a LCDR3 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 15, 31, 47,63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287,303, 319, 335, 351, 367, and 383, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%homology thereof.

The present invention also provides an antibody or fragment thereofwhich further comprises a HCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83,99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307,323, 339, 355, and 371, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof;a HCDR2 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165,181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357, and 373, ora substantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof; a LCDR1 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 11,27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219, 235, 251,267, 283, 299, 315, 331, 347, 363, and 379, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof; and a LCDR2 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 13, 29, 45,61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285,301, 317, 333, 349, 365, and 381, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%homology thereof.

According to certain embodiments, the antibody or fragment thereofcomprises the heavy and light chain CDR sequences encoded by the nucleicacid sequences of SEQ ID NOs: 1 and 9 (e.g. H1M085N), 17 and 25 (e.g.H1M086N), 33 and 41 (e.g. H1M089N), 49 and 57 (e.g. H1M102N), 65 and 73(e.g. H1M103N), 81 and 89 (e.g. H1M116N), 97 and 105 (e.g. H1H134P), 113and 121 (e.g. H1H136P), 129 and 137 (e.g. H1H141P), 145 and 153 (e.g.H1H142P), 161 and 169 (e.g. H1H143P), 177 and 185 (e.g. H1H144P), 193and 201 (e.g. H1H145P), 209 and 217 (e.g. H1H147P), 225 and 233 (e.g.H1H151P), 241 and 249 (e.g. H1H153P), 257 and 265 (e.g. H1H155P), 273and 281 (e.g. H1H157P), 289 and 297 (e.g. H1H158P), 305 and 313 (e.g.H1H159P), 321 and 329 (e.g. H1H161P), 337 and 345 (e.g. H1H163P), 353and 361 (e.g. H1H169P), or 369 and 377 (e.g. H1H171P).

The present invention includes anti-EGFR antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds EGFR and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-EGFR antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-EGFR antibody. Exemplary agentsthat may be advantageously combined with an anti-EGFR antibody include,without limitation, other agents that inhibit EGFR activity (includingother antibodies or antigen-binding fragments thereof, peptideinhibitors, small molecule antagonists, etc) and/or agents which do notdirectly bind EGFR but nonetheless interfere with, block or attenuateEGFR-mediated signaling.

In yet another aspect, the invention provides methods for inhibitingEGFR activity using an anti-EGFR antibody or antigen-binding portion ofan antibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment of anantibody of the invention. The disorder treated is any disease orcondition which is improved, ameliorated, inhibited or prevented byremoval, inhibition or reduction of EGFR activity. The anti-EGFRantibodies or antibody fragments of the invention may function to blockthe interaction between EGFR and an EGFR binding partner (e.g.,epidermal growth factor [EGF], transforming growth factor-alpha [TGF-α],etc.), or otherwise inhibit the signaling activity of EGFR.

The present invention also includes the use of an anti-EGFR antibody orantigen binding portion of an antibody of the invention in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by EGFR activity in a patient.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

DEFINITIONS

The expressions “EGFR” and “EGFR fragment,” as used herein refer to thehuman EGFR protein or fragment unless specified as being from anon-human species (e.g., “mouse EGFR,” “mouse EGFR fragment,” “monkeyEGFR,” “monkey EGFR fragment,” etc.). The extracellular domain of humanEGFR has the amino acid sequence shown in, e.g., amino acids 25 to 645of SEQ ID NO:385.

As used herein, “an antibody that binds EGFR” or an “anti-EGFR antibody”includes antibodies, and antigen-binding fragments thereof, that bind asoluble fragment of an EGFR protein (e.g., a portion of theextracellular domain of EGFR) and/or cell surface-expressed EGFR. Theexpression “cell surface-expressed EGFR” means an EGFR protein orportion thereof that is/are expressed on the surface of a cell in vitroor in vivo, such that at least a portion of the EGFR protein (e.g.,amino acids 25 to 645 of SEQ ID NO:385) is exposed to the extracellularside of the cell membrane and accessible to an antigen-binding portionof an antibody. Soluble EGFR molecules include, e.g., monomeric anddimeric EGFR constructs as described in Example 3 herein (i.e.,“EGFR.mmh”, SEQ ID NO:386, and “EGFR.mFc”, SEQ ID NO:387, respectively),or constructs substantially similar thereto.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., EGFR). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-EGFR antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3, (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2, (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to EGFR: (i) interferes with theinteraction between EGFR or an EGFR fragment and an EGFR ligand (e.g.,EGF, TGF-α, etc.), and/or (ii) results in inhibition of at least onebiological function of EGFR. The inhibition caused by an EGFRneutralizing or blocking antibody need not be complete so long as it isdetectable using an appropriate assay. Exemplary assays for detectingEGFR inhibition are described herein.

The anti-EGFR antibodies disclosed herein may comprise one or more aminoacid substitutions, insertions and/or deletions in the framework and/orCDR regions of the heavy and light chain variable domains as compared tothe corresponding germline sequences from which the antibodies werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-EGFR antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-EGFR antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Biological Characteristics of the Antibodies

The present invention includes anti-EGFR antibodies and antigen-bindingfragments thereof that bind monomeric or dimeric EGFR molecules withhigh affinity. For example, the present invention includes antibodiesand antigen-binding fragments of antibodies that bind dimeric EGFR witha K_(D) of less than about 20 pM as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind dimeric EGFR with a K_(D) of less than about15 pM, less than about 10 pM, less than about 8 pM, less than about 6pM, less than about 4 pM, less than about 2 pM, or less than about 1 pM,as measured by surface plasmon resonance, e.g., using the assay formatas defined in Example 3 herein. The present invention also includesanti-EGFR antibodies and antigen-binding fragments thereof that binddimeric EGFR with a t½ of greater than about 200 minutes as measured bysurface plasmon resonance, e.g., using the assay format as defined inExample 3 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind dimeric EGFRwith a t½ of greater than about 210 minutes, greater than about 220minutes, greater than about 250 minutes, greater than about 260 minutes,greater than about 280 minutes, greater than about 300 minutes, greaterthan about 320 minutes, greater than about 340 minutes, greater thanabout 360 minutes, greater than about 380 minutes, greater than about400 minutes, greater than about 450 minutes, greater than about 500minutes, greater than about 550 minutes, greater than about 600 minutes,greater than about 650 minutes, greater than about 800 minutes, greaterthan about 1000 minutes, or more, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein.

The present invention also includes anti-EGFR antibodies andantigen-binding fragments thereof that inhibit the growth ofEGFR-expressing tumor cells. For example, the present invention includesanti-EGFR antibodies and antigen-binding fragments thereof that inhibitthe growth of tumor cells that express high levels of EGFR on theirsurface (e.g., A431 epidermoid carcinoma cells), with an IC₅₀ (i.e., theconcentration resulting in 50% maximal growth inhibition) of less thanabout 200 pM. IC₅₀ values can be determined using the cell growthinhibition assay exemplified in Example 4 herein, or a substantiallysimilar assay. According to certain embodiments of the present inventionthe anti-EGFR antibodies or antigen-binding fragments thereof are ableto inhibit the growth of A431 cells in vitro with an IC₅₀ of less thanabout 180 pM, less than about 160 pM, less than about 140 pM, less thanabout 120 pM, less than about 100 pM, less than about 80 pM, less thanabout 60 pM, less than about 40 pM, less than about 20 pM, less thanabout 10 pM, less than about 5 pM, or less than about 2 pM, asdetermined using the cell growth inhibition assay exemplified in Example4 herein, or a substantially similar assay.

The present invention also includes anti-EGFR antibodies andantigen-binding fragments thereof that induce antibody-dependentcell-mediated cytotoxicity (ADCC) of cells that express EGFR. Assays formeasuring ADCC are known in the art. An exemplary assay format isillustrated in Example 5 herein, in which anti-EGFR antibodies are addedto a cellular mixture of peripheral blood mononuclear cells (PBMCs) andA431 epidermoid carcinoma cells (i.e., cells expressing high levels ofEGFR). The extent of cell killing is assessed relative to the maximalcell lysis signal observed under conditions in which untreated cellswere lysed by addition of digitonin; the extent of ADCC can thereby beexpressed in terms of the percent of maximum cell killing. The presentinvention includes anti-EGFR antibodies that produce a maximum cellkilling percentage of greater than about 25%, when tested in the ADCCassay format of Example 5, or a substantially similar assay. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention induce ADCC with a maximum cell killing percentage of about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, or more, as measured in the ADCC assayformat of Example 5 or a substantially similar assay.

The present invention also includes anti-EGFR antibodies andantigen-binding fragments thereof that inhibit tumor growth in vitro orin vivo. In certain circumstances, the antibodies or antigen-bindingfragments of the present invention cause tumor regression or shrinkage.The present invention includes anti-EGFR antibodies and antigen-bindingfragments thereof that inhibit the growth of human tumor xenografts inimmunocompromised mice. For example, as illustrated in Example 6 herein,the exemplary anti-EGFR antibody H1H141P significantly inhibited thegrowth of head and neck squamous cell carcinoma cells (e.g., FaDu tumorcells), pancreatic tumor cells (BxPC3), and lung tumor cells (Calu3 andNCI-H358), in mouse xenografts models. The invention includes antibodiesand antigen-binding fragments thereof that inhibit tumor cell growth intumor-bearing mice by greater than about 50% (e.g., about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or more) as compared to hFc-control-treated tumor-bearingmice.

The present invention also includes anti-EGFR antibodies and antigenbinding fragments thereof that induce internalization of cell surfaceexpressed EGFR.

Epitope Mapping and Related Technologies

The present invention includes anti-EGFR antibodies which interact withone or more amino acids found within the extracellular domain of humanEGFR (e.g., within extracellular domain I, II, III, and/or IV). Theepitope to which the antibodies bind may consist of a single contiguoussequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) amino acids located within theextracellular domain of EGFR. Alternatively, the epitope may consist ofa plurality of non-contiguous amino acids (or amino acid sequences)located within the extracellular domain of EGFR.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-EGFR antibodies that bind tothe same epitope as any of the specific exemplary antibodies describedherein (e.g. H1M085N, H1M086N, H1M089N, H1M102N, H1M103N, H1M116N,H1H134P, H1H136P, H1H141P, H1H142P, H1H143P, H1H144P, H1H145P, H1H147P,H1H151P, H1H153P, H1H155P, H1H157P, H1H158P, H1H159P, H1H161P, H1H163P,H1H169P, H1H171P etc.). Likewise, the present invention also includesanti-EGFR antibodies that compete for binding to EGFR with any of thespecific exemplary antibodies described herein (e.g. H1M085N, H1M086N,H1M089N, H1M102N, H1M103N, H1M116N, H1H134P, H1H136P, H1H141P, H1H142P,H1H143P, H1H144P, H1H145P, H1H147P, H1H151P, H1H153P, H1H155P, H1H157P,H1H158P, H1H159P, H1H161P, H1H163P, H1H169P, H1H171P etc.).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-EGFR antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-EGFRantibody of the invention, the reference antibody is allowed to bind toan EGFR protein (e.g., a soluble portion of the EGFR extracellulardomain or cell surface-expressed EGFR). Next, the ability of a testantibody to bind to the EGFR molecule is assessed. If the test antibodyis able to bind to EGFR following saturation binding with the referenceanti-EGFR antibody, it can be concluded that the test antibody binds toa different epitope than the reference anti-EGFR antibody. On the otherhand, if the test antibody is not able to bind to the EGFR moleculefollowing saturation binding with the reference anti-EGFR antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference anti-EGFR antibody of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, Biacore, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present invention, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20- or 100-fold excess of one antibody inhibits binding of theother by at least 50% but preferably 75%, 90% or even 99% as measured ina competitive binding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding with a referenceanti-EGFR antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to an EGFR protein (e.g., a soluble portion of the EGFRextracellular domain or cell surface-expressed EGFR) under saturatingconditions followed by assessment of binding of the test antibody to theEGFR molecule. In a second orientation, the test antibody is allowed tobind to an EGFR molecule under saturating conditions followed byassessment of binding of the reference antibody to the EGFR molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the EGFR molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to EGFR. Aswill be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the same epitope as the reference antibody, but maysterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human EGFR.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to EGFR areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-EGFR antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies but that retain the ability to bind human EGFR.Such variant antibodies and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-EGFRantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-EGFR antibody or antibody fragment that isessentially bioequivalent to an anti-EGFR antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-EGFR antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-EGFR antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-EGFRantibodies bind to human EGFR but not to EGFR from other species. Thepresent invention also includes anti-EGFR antibodies that bind to humanEGFR and to EGFR from one or more non-human species. For example, theanti-EGFR antibodies of the invention may bind to human EGFR and maybind or not bind, as the case may be, to one or more of mouse, rat,guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow,horse, camel, cynomologous, marmoset, rhesus or chimpanzee EGFR.

Immunoconjugates

The invention encompasses anti-EGFR monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-EGFR antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human EGFR or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-EGFR antibodies or antigen-binding fragments thereof of the presentinvention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with EGFR activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-EGFRantibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by EGFR expression or activity, or treatableby blocking the interaction between EGFR and an EGFR ligand (e.g., EGFor TGF-α) or otherwise inhibiting EGFR activity and/or signaling, and/orpromoting receptor internalization and/or decreasing cell surfacereceptor number. For example, the antibodies and antigen-bindingfragments of the present invention are useful for the treatment oftumors that express high levels of EGFR. The antibodies andantigen-binding fragments of the present invention may be used to treat,e.g., primary and/or metastatic tumors arising in the brain andmeninges, oropharynx, lung and bronchial tree, gastrointestinal tract,male and female reproductive tract, muscle, bone, skin and appendages,connective tissue, spleen, immune system, blood forming cells and bonemarrow, liver and urinary tract, and special sensory organs such as theeye. In certain embodiments, the antibodies and antigen-bindingfragments of the invention are used to treat one or more of thefollowing cancers: renal cell carcinoma, pancreatic carcinoma, breastcancer, head and neck cancer, prostate cancer, malignant gliomas,osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancerwith MET amplification), malignant mesothelioma, multiple myeloma,ovarian cancer, small cell lung cancer, non-small cell lung cancer(e.g., EGFR-dependent non-small cell lung cancer), synovial sarcoma,thyroid cancer, or melanoma.

Combination Therapies and Formulations

The present invention includes therapeutic administration regimens whichcomprise administering an anti-EGFR antibody of the present invention incombination with at least one additional therapeutically activecomponent. Non-limiting examples of such additional therapeuticallyactive components include other EGFR antagonists (e.g., a secondanti-EGFR antibody [e.g., cetuximab or panitumumab] or small moleculeinhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist ofanother EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g.,anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small moleculeinhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII(e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist(e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1Rantibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879,PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), aPDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody), a VEGF antagonist(e.g., a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,411 (also referred toherein as a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), etc. Other agents that may bebeneficially administered in combination with the anti-EGFR antibodiesof the invention include cytokine inhibitors, including small-moleculecytokine inhibitors and antibodies that bind to cytokines such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17,IL-18, or to their respective receptors.

The present invention also includes therapeutic combinations comprisingany of the anti-EGFR antibodies mentioned herein and an inhibitor of oneor more of VEGF, Ang2, DLL4, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R,B-raf, PDGFR-α, PDGFR-β, or any of the aforementioned cytokines, whereinthe inhibitor is an aptamer, an antisense molecule, a ribozyme, ansiRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fabfragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAbfragment; or other engineered molecules, such as diabodies, triabodies,tetrabodies, minibodies and minimal recognition units). The anti-EGFRantibodies of the invention may also be administered and/orco-formulated in combination with antivirals, antibiotics, analgesics,corticosteroids and/or NSAIDs. The anti-EGFR antibodies of the inventionmay also be administered as part of a treatment regimen that alsoincludes radiation treatment and/or conventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan anti-EGFR antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-EGFR antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-EGFR antibody ofthe present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

The present invention also includes compositions and methods comprisinga combination of a “degrading antibody” and a “ligand-blockingantibody.” A “degrading antibody” means an anti-EGFR antibody thatcauses degradation of EGFR in cells without necessarily blockingligand-receptor interactions. A non-limiting example of a degradingantibody of the present invention is the antibody designated H1H134P. A“ligand-blocking antibody” means an anti-EGFR antibody that blocks theinteraction between EGFR and one or more of its ligands (e.g., EGF orTGF-α). A non-limiting example of a ligand-blocking antibody of thepresent invention is the antibody designated H1H141P. Another example ofa ligand blocking antibody is cetuximab. The present inventors haveconceived of combining a degrading antibody and a ligand-blockingantibody in order to synergistically or otherwise improve anti-tumorefficacy. Accordingly, the present invention includes pharmaceuticalcompositions comprising at least one degrading antibody and at least oneligand-blocking antibody. The present invention also includestherapeutic methods comprising administering to a subject a combinationof a degrading antibody and a ligand-blocking antibody (either asseparate administrations or as co-formulations).

Diagnostic Uses of the Antibodies

The anti-EGFR antibodies of the present invention may also be used todetect and/or measure EGFR, or EGFR-expressing cells in a sample, e.g.,for diagnostic purposes. For example, an anti-EGFR antibody, or fragmentthereof, may be used to diagnose a condition or disease characterized byaberrant expression (e.g., over-expression, under-expression, lack ofexpression, etc.) of EGFR. Exemplary diagnostic assays for EGFR maycomprise, e.g., contacting a sample, obtained from a patient, with ananti-EGFR antibody of the invention, wherein the anti-EGFR antibody islabeled with a detectable label or reporter molecule. Alternatively, anunlabeled anti-EGFR antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure EGFR in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in EGFR diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of EGFR protein, orfragments thereof, under normal or pathological conditions. Generally,levels of EGFR in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal EGFR levels or activity) will be measured to initiallyestablish a baseline, or standard, level of EGFR. This baseline level ofEGFR can then be compared against the levels of EGFR measured in samplesobtained from individuals suspected of having a EGFR related disease orcondition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to EGFR

An EGFR-expressing cell line was administered directly, with an adjuvantto stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNAencoding human Immunoglobulin heavy and kappa light chain variableregions. The antibody immune response was monitored by a EGFR-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce EGFR-specificantibodies. Using this technique several anti-EGFR chimeric antibodies(i.e., antibodies possessing human variable domains and mouse constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H1M085N, H1M086N, H1M089N, H1M102N, H1M103N,and H1M116N.

Anti-EGFR antibodies were also isolated directly from antigen-positive Bcells without fusion to myeloma cells, as described in US2007/0280945A1. Using this method, several fully human anti-EGFRantibodies (i.e., antibodies possessing human variable domains and humanconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as follows: H1H134P, H1H136P, H1H141P, H1H142P,H1H143P, H1H144P, H1H145P, H1H147P, H1H151P, H1H153P, H1H155P, H1H157P,H1H158P, H1H159P, H1H161P, H1H163P, H1H169P, and H1H171P.

Certain biological properties of the exemplary anti-EGFR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-EGFR antibodies and their correspondingantibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 085N 2 4 6 8 10 12 14 16 086N 18 20 22 24 26 28 30 32089N 34 36 38 40 42 44 46 48 102N 50 52 54 56 58 60 62 64 103N 66 68 7072 74 76 78 80 116N 82 84 86 88 90 92 94 96 134P 98 100 102 104 106 108110 112 136P 114 116 118 120 122 124 126 128 141P 130 132 134 136 138140 142 144 142P 146 148 150 152 154 156 158 160 143P 162 164 166 168170 172 174 176 144P 178 180 182 184 186 188 190 192 145P 194 196 198200 202 204 206 208 147P 210 212 214 216 218 220 222 224 151P 226 228230 232 234 236 238 240 153P 242 244 246 248 250 252 254 256 155P 258260 262 264 266 268 270 272 157P 274 276 278 280 282 284 286 288 158P290 292 294 296 298 300 302 304 159P 306 308 310 312 314 316 318 320161P 322 324 326 328 330 332 334 336 163P 338 340 342 344 346 348 350352 169P 354 356 358 360 362 364 366 368 171P 370 372 374 376 378 380382 384

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H” or “H1M”), followed by a numericalidentifier (e.g. “085” or “134” as shown in Table 1), followed by a “P”or “N” suffix. Thus, according to this nomenclature, an antibody may bereferred to herein as, e.g., “H1M085N” or “H1H134P”, etc. The H1H andH1M prefixes on the antibody designations used herein indicate theparticular Fc region of the antibody. For example, an “H1M” antibody hasa mouse IgG1 Fc, whereas an “H1H” antibody has a human IgG1 Fc. As willbe appreciated by a person of ordinary skill in the art, an H1M antibodycan be converted to an H1H antibody, and vice versa, but in any event,the variable domains (including the CDRs)—which are indicated by thenumerical identifiers shown in Table 1—will remain the same.

Control Constructs Used in the Following Examples

Various control constructs (anti-EGFR antibodies) were included in thefollowing experiments for comparative purposes. The control constructsare designated as follows: Control I: a chimeric anti-EGFR antibody withheavy and light chain variable sequences of “mAb 225” as set forth inU.S. Pat. No. 7,060,808; and Control II: a commercially available fullyhuman monoclonal anti-EGFR antibody designated as ABX-EGF, also known asPanitumumab or Vectibix®.

Example 3 Antibody Binding to Human EGFR as Determined by SurfacePlasmon Resonance

Equilibrium dissociation constants (K_(D) values) for antigen binding toselected purified anti-human EGFR monoclonal antibodies were determinedusing a real-time surface plasmon resonance biosensor (Biacore T100)assay at 37° C. The Biacore sensor surface was derivatized withmonoclonal mouse anti-human Fc antibody (GE Biosciences) to captureanti-EGFR monoclonal antibodies, expressed in the human IgG1 Fc format(antibody prefix H1H). Different concentrations of human monomeric(EGFR.mmh; SEQ ID NO:386) and dimeric (EGFR.mFc; SEQ ID NO:387) proteinswere injected over the anti-EGFR monoclonal antibody captured surface ata flow rate of 50 μl/min. Antibody-antigen association was monitored for4-5 minutes while dissociation of antigen from the captured monoclonalantibody surface was monitored for 10 min. Kinetic association (k_(a))and dissociation (k_(d)) rate constants were determined by processingand fitting the data to a 1:1 binding model using Scrubber 2.0 curvefitting software. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t_(1/2)) were calculated from the kinetic rateconstants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=(In2/(60*k_(d)).Kinetic binding parameters for different anti-EGFR monoclonal antibodiesare shown in Table 2.

TABLE 2 Binding Characteristics of Anti-EGFR Antibodies to Monomeric andDimeric EGFR ka kd K_(D) t_(1/2) Antibody Analyte (Ms⁻¹) (s⁻¹) (Molar)(min) H1H0085N EGFR.mmh 1.19E+05 1.85E−03 1.55E−08 6 EGFR.mFc 3.36E+054.05E−05 1.20E−10 286 H1H086N EGFR.mmh 5.40E+05 7.08E−04 1.31E−09 16EGFR.mFc 1.90E+06 1.13E−05 5.98E−12 1018 H1H089N EGFR.mmh 3.13E+056.83E−03 2.18E−08 2 EGFR.mFc 1.49E+06 4.49E−05 3.01E−11 257 H1H102NEGFR.mmh 1.66E+05 9.43E−04 5.67E−09 12 EGFR.mFc 8.63E+05 5.62E−056.51E−11 206 H1H103N EGFR.mmh 1.00E+05 1.39E−03 1.39E−08 8 EGFR.mFc3.83E+05 4.19E−05 1.09E−10 276 H1H116N EGFR.mmh 5.39E+05 2.84E−035.27E−09 4 EGFR.mFc 1.55E+06 3.16E−05 2.03E−11 366 H1H134P EGFR.mmh9.30E+05 7.89E−04 8.48E−10 15 EGFR.mFc 3.09E+06 2.47E−05 7.99E−12 468H1H136P EGFR.mmh NB NB NB NB EGFR.mFc NB NB NB NB H1H141P EGFR.mmh3.96E+05 4.05E−04 1.02E−09 29 EGFR.mFc 9.03E+05 7.51E−06 8.31E−12 1539H1H142P EGFR.mmh 1.58E+05 6.89E−04 4.35E−09 17 EGFR.mFc 3.61E+051.20E−05 3.32E−11 965 H1H143P EGFR.mmh 1.27E+05 7.27E−04 5.71E−09 16EGFR.mFc 3.81E+05 1.35E−05 3.54E−11 856 H1H144P EGFR.mmh 1.84E+059.67E−04 5.25E−09 12 EGFR.mFc 8.94E+05 1.94E−05 2.17E−11 596 H1H145PEGFR.mmh 1.37E+05 1.95E−04 1.43E−09 59 EGFR.mFc 2.52E+05 5.86E−052.32E−10 197 H1H147P EGFR.mmh 6.54E+04 3.76E−04 5.76E−09 31 EGFR.mFc1.90E+05 1.26E−05 6.64E−11 914 H1H151P EGFR.mmh 1.34E+05 1.13E−038.40E−09 10 EGFR.mFc 1.34E+05 1.13E−03 8.40E−09 10 H1H153P EGFR.mmh7.61E+04 2.35E−04 3.09E−09 49 EGFR.mFc 2.34E+05 8.23E−06 3.51E−11 1403H1H155P EGFR.mmh 1.76E+05 1.69E−04 9.62E−10 68 EGFR.mFc 3.29E+051.06E−04 3.21E−10 109 H1H157P EGFR.mmh NB NB NB NB EGFR.mFc NB NB NB NBH1H158P EGFR.mmh 1.31E+05 7.73E−04 5.90E−09 15 EGFR.mFc 4.08E+059.84E−06 2.41E−11 1174 H1H159P EGFR.mmh 4.76E+05 3.25E−04 6.82E−10 36EGFR.mFc 1.64E+06 5.63E−06 3.44E−12 2051 H1H161P EGFR.mmh 4.89E+053.21E−04 6.55E−10 36 EGFR.mFc 1.73E+06 2.76E−06 1.59E−12 4187 H1H163PEGFR.mmh 5.11E+05 4.26E−04 8.34E−10 27 EGFR.mFc 1.81E+06 2.12E−061.17E−12 5447 H1H169P EGFR.mmh 6.65E+05 8.69E−04 1.31E−09 13 EGFR.mFc2.29E+06 1.69E−05 7.36E−12 684 H1H171P EGFR.mmh 7.94E+04 1.13E−031.42E−08 10 EGFR.mFc 3.39E+05 3.48E−05 1.03E−10 332 Control I EGFR.mmh1.58E+06 7.38E−03 4.68E−09 2 EGFR.mFc 3.55E+06 1.08E−04 3.03E−11 107Control II EGFR.mmh 7.12E+05 7.62E−04 1.07E−09 15 EGFR.mFc 1.38E+065.82E−05 4.23E−11 198

As shown in Table 2, several of the anti-EGFR antibodies of the presentinvention exhibited superior binding properties as compared to thecontrol antibodies. For example, certain anti-EGFR antibodies of thepresent invention exhibited K_(D) values less than 10 pM and t½ valuesgreater than 400 minutes, when tested for binding to dimeric EGFR(“EGFR.mFc”) in the surface plasmon resonance assay described above;e.g., H1H086N (5.98 pM/1018 min), H1H134P (7.99 pM/468 min), H1H141P(8.31 pM/1539 min), H1H159P (3.44 pM/2059 min), H1H161P (1.59 pM/4187min), and H1H169P (7.36 pM/684 min). By contrast, Control I exhibited aK_(D) of 30.3 pM and a t½ of 107 min, and Control II exhibited a K_(D)of 42.3 pM and a t½ of 198 min when tested for binding to dimeric EGFRunder identical experimental conditions.

Example 4 Inhibition of Cell Growth by Anti-EGFR Antibodies

Anti-EGFR antibodies were tested for their ability to inhibit the growthof A431 epidermoid carcinoma cells in vitro. A431 cells have anamplification of the EGFR gene and exhibit a strong dependence on EGFRsignaling for growth. A431 cells were seeded at a density of 5.0×10³cells per well in 96-well plates and incubated in DMEM medium containing0.5% BSA and 1% penicillin/streptomycin/glutamine. EGFR-specific mAbswere added to cells in 1:3 serial dilutions starting at 60 nM. After 7days viable cells were quantified by staining with Alamar Blue(Invitrogen) and measuring fluorescence with a Flexstation IIIspectrophotometer. Absorbance values were plotted using a four-parameterlogistic equation over the 12-point dilution series (GraphPad Prism).Results are summarized in Table 3.

TABLE 3 Relative Inhibition of A431 Cell Proliferation by Anti-EGFRAntibodies IC₅₀ of A431 Proliferation Antibody (Molar) H1H0085N1.645E−09 H1H086N 5.459E−10 H1H089N 2.305E−09 H1H102N 2.883E−10 H1H103N 3.89E−09 H1H116N  2.24E−10 H1H134P 1.844E−09 H1H136P No InhibitionH1H141P 1.405E−10 H1H142P 5.474E−10 H1H143P 9.968E−11 H1H144P 1.393E−11H1H145P No Inhibition H1H147P 9.769E−11 H1H151P  2.77E−10 H1H153P NoInhibition H1H155P No Inhibition H1H157P No Inhibition H1H158P NoInhibition H1H159P No Inhibition H1H161P No Inhibition H1H163P NoInhibition H1H169P No Inhibition H1H171P No Inhibition Control I3.346E−10

As shown in Table 3, the tested antibodies exhibited a broad range ofIC₅₀ values with some antibodies possessing little to no blockingactivity while others displayed IC₅₀ values lower than the referenceControl I antibody. For example, anti-EGFR antibodies H1H141P, H1H143P,H1H144P and H1H147P all exhibited IC₅₀ values less than 200 pM, whereas,the Control I antibody exhibited an IC₅₀ value of greater than 334 pM.

Example 5 Induction of ADCC on A431 Cells by Anti-EGFR Antibodies

Antibody dependent cell-mediated cytotoxicity (ADCC) is a cellularprocess which occurs when Fc receptors on natural killer cells areactivated to induce the release of cell-lysing enzymes against targetcells. The ability of anti-EGFR antibodies to induce ADCC in vitro wasassessed by using peripheral blood mononuclear cells (PBMC) as effector,or primary “killer” cells, against A431 target cells that endogenouslyover-express EGFR.

Briefly, anti-EGFR antibodies over a broad concentration range (40 nM-0nM; 1:3 dilutions) were added to a cellular mixture of PBMC and A431cells (30:1 ratio) in 96-well plates. The plates were incubated for 4hours at 37° C., 5% CO₂, equilibrated to room temperature for 10 minutesand CytoTox-Glo reagent was added to the wells. Untreated cells incontrol wells were lysed by addition of digitonin to determine maximalcell lysis signal. The plates were incubated briefly at room temperatureand luminescence was measured from each well using a plate reader.

Cytotoxic response was calculated by subtracting the signal for A431cells incubated with PBMC without the addition of anti-EGFR antibodies(background) from the signal generated from target cells mixed with PBMCin the presence of anti-EGFR mAbs. The percentage of cytotoxicity wascalculated by dividing the cytotoxic response of cells againstbackground by the maximal cytotoxic response obtained from cell lysisvia digitonin. Data was analyzed by a four-parameter logistical equationwith a sigmoidal dose-response curve (Graph Pad Prism). Results aresummarized in Table 4. (NA=no activity).

TABLE 4 ADCC Activity of Selected Anti-EGFR Antibodies Maximum CellAntibody EC₅₀ (Molar) Killing (%) H1H0085N 3.70E−13 35 H1H086N 2.91E−1325 H1H089N 5.83E−12 53 H1H102N 4.39E−13 48 H1H103N 7.25E−13 17 H1H116N2.51E−13 29 H1H134P 4.61E−11 33 H1H136P 1.49E−09 27 H1H141P 2.78E−12 41H1H142P 1.19E−12 34 H1H143P NA 13 H1H144P 4.57E−11 43 H1H145P NA 0H1H147P NA 13 H1H151P 5.48E−09 68 H1H153P 2.65E−11 31 H1H155P 1.67E−1130 H1H157P 2.27E−13 36 H1H158P 2.32E−12 28 H1H159P 2.69E−09 60 H1H161P9.80E−12 38 H1H163P 2.96E−10 48 H1H169P 4.81E−09 27 H1H171P 8.94E−13 27Control I 1.86E−12 24

As shown in Table 4, several anti-EGFR mAbs induced ADCC on A431 cellsco-incubated with PBMC effector cells. Additionally, several anti-EGFRmAbs demonstrate a high maximal cell killing percentage comparable tothe control antibody (Control I). For example, anti-EGFR antibodiesH1H089N, H1H102N, H1H141P, H1H144P, H1H151P, H1H159P and H1H163P eachexhibited greater than 40% maximum cell killing in the ADCC assay,whereas the Control I antibody exhibited less than 25% maximum cellkilling in this assay.

Example 6 Inhibition of Tumor Growth by an Anti-EGFR Antibody

The anti-EGFR antibody H1H141P was tested for its ability to inhibit thegrowth human tumor xenografts in immunocompromised mice. Briefly, 2×10^6FaDu head and neck squamous cell carcinoma cells were implantedsubcutaneously into the flank of C.B.-17 SCID mice. After tumors reachedan average size of approximately 200 mm³ mice were randomized intogroups for treatment (N=6 mice per group) and injected twice per weeksubcutaneously with either human Fc control protein (12.5 mg/kg; SEQ IDNO:388) or with H1H141P (10 mg/kg). Mice were treated for 15 days.

Tumor volumes were measured twice per week throughout the experiment andtumor weights were determined upon excision at the conclusion of theexperiment. The average tumor growth (the average change in tumor volumefrom the start of treatment through the end of the experiment) and theaverage tumor weights were determined for each group. Results aresummarized in Table 5.

TABLE 5 Inhibition of FaDu Tumor Growth in SCID Mice Tumor Growth % De-% De- (mm³) crease in crease in from start Tumor Tumor Tumor Antibody oftreatment Growth vs Weight (g) Weight vs (mg/kg) (mean ± SD) Control(mean ± SD) Control hFc Control 1099 ± 186 — 0.993 ± 0.176 — (12.5)H1H141P  55 ± 115 95 0.215 ± 0.120 78 (10)

In a similar experiment, the effect of H1H141P on the growth of BxPC3pancreatic tumor xenografts was determined, as summarized in Table 6.

TABLE 6 Inhibition of BxPC3 Tumor Growth in SCID Mice Tumor Growth % De-% De- (mm³) crease in crease in from start Tumor Tumor Tumor Antibody oftreatment Growth vs Weight (g) Weight vs (mg/kg) (mean ± SD) Control(mean ± SD) Control hFc Control 706 ± 277 — 0.926 ± 0.412 — (25) H1H141P97 ± 59 86 0.275 ± 0.098 70 (12.5)

In a similar experiment, the effect of H1H141P on the growth of Calu3lung tumor xenografts was determined, as summarized in Table 7.

TABLE 7 Inhibition of Calu3 Tumor Growth in SCID Mice Tumor Growth % De-% De- (mm³) crease in crease in from start Tumor Tumor Tumor Antibody oftreatment Growth vs Weight (g) Weight vs (mg/kg) (mean ± SD) Control(mean ± SD) Control hFc Control 656 ± 202 — 0.884 ± 0.275 — (25) H1H141P335 ± 58  49 0.582 ± 0.097 34 (25)

In a similar experiment, the effect of H1H141P on the growth of NCI-H358lung tumor xenografts was determined, as summarized in Table 8.

TABLE 8 Inhibition of NCI-H358 Tumor Growth in SCID Mice Tumor Growth(mm³) from start % Decrease in Antibody of treatment Tumor Growth vs(mg/kg) (mean ± SD) Control hFc Control (25)   329 ± 170 — H1H141P(12.5) (−14) ± 47 104

In a similar experiment, the effect of H1H141P on the growth of A431epidermoid carcinoma xenografts was determined, as summarized in Table9.

TABLE 9 Inhibition of A431 Tumor Growth in SCID Mice Tumor Growth (mm³)from start % Decrease in Antibody of treatment Tumor Growth vs (mg/kg)(mean ± SD) Control hFc Control (25) 134 ± 173 — H1H141P (12.5) 62 ± 4995 H1H141P (25) 45 ± 60 97

Collectively, these findings indicate that H1H141P as a monotherapy caninhibit the growth of multiple human tumor xenografts, representingseveral different tumor types.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody, or antigen-binding fragmentthereof, that specifically binds human epidermal growth factor receptor(hEGFR), wherein the antibody or antigen-binding fragment comprises: (a)the complementarity determining regions (CDRs) of a heavy chain variableregion (HCVR) having an amino acid sequence of SEQ ID NO: 130 and (b)the CDRs of a light chain variable region (LCVR) having an amino acidsequence of SEQ ID NO:
 138. 2. The isolated antibody or antigen-bindingfragment of claim 1, wherein the antibody or antigen-binding fragmentcomprises the heavy and light chain CDRs of a HCVR/LCVR amino acidsequence pair having SEQ ID NOs: 130/138.
 3. The isolated antibody orantigen-binding fragment of claim 2, wherein the antibody orantigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, having SEQ ID NOs: 132-134-136-140-142-144.
 4. Apharmaceutical composition comprising the antibody or antigen-bindingfragment of claim 1, and a pharmaceutically acceptable carrier ordiluent.